This invention relates to nucleic acid and amino acid sequences of oxidoreductase molecules and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative disorders including cancer, endocrine, metabolic, reproductive, neurological, autoimmune/inflammatory, and viral disorders.
Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to the reduction or oxidation of a donor or acceptor cofactor. Potential cofactors include cyrochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E. A. and Leech, A. R. (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U. K. pp. 779-793).
Reductase activity catalyzes the transfer of electrons between substrate(s) and cofactor(s) with concurrent oxidation of the cofactor. The reverse dehydrogenase reaction catalyzes the reduction of a cofactor and consequent oxidation of the substrate. Oxidoreductase enzymes are a broad superfamily of proteins that catalyze numerous reactions in all cells of organisms ranging from bacteria to plants to humans. These reactions include metabolism of sugar, certain detoxification reactions in the liver, and the synthesis or degradation of fatty acids, amino acids, glucocorticoids, estrogens, androgens, and prostaglandins. Different family members are named according to the direction in which their reactions are typically catalyzed; thus they may be referred to as oxidoreductases, oxidases, reductases, or dehydrogenases. In addition, family members often have distinct cellular localizations, including the cytosol, the plasma membrane, mitochondrial inner or outer membrane, and peroxisomes.
Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase, which includes three enzyme activities: tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. Thus, tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells.
Intracellular redox status plays a critical role in the assembly of proteins. A major rate limiting step in protein folding is the thiol:disulfide exchange necessary for correct protein assembly. Although incubation of reduced, unfolded proteins in buffers containing defined ratios of oxidized and reduced thiols can lead to folding into native conformation, the rate-of folding is slow, and the attainment of the native conformation decreases proportionately with protein size and the number of cysteine residues. Certain cellular compartments such as the endoplasmic reticulum of eukaryotes and the periplasmic space of prokaryotes are maintained in a more oxidized state than the surrounding cytosol. Correct disulfide formation can occur in these compartments, but it occurs at a rate that is insufficient for normal cell processes and inadequate for synthesizing secreted proteins.
Protein disulfide isomerases (PDIs), thioredoxins, and glutaredoxins are able to catalyze the formation of disulfide bonds and regulate the redox environment in cells to enable the necessary thiol:disulfide exchanges. Each of these classes of molecules has a somewhat different function, but all belong to a group of disulfide-containing redox proteins that contain a conserved active-site sequence and are ubiquitously distributed in eukaryotes and prokaryotes. PRIs are found in the endoplasmic reticulum of eukaryotes and in the periplasmic space of prokaryotes. PRIs function by exchanging their own disulfide for thiols in a folding peptide chain. In contrast, reduced thioredoxins and glutaredoxins are generally found in the cytoplasm and function by directly reducing disulfides in the substrate proteins. Thioredoxin (Trx), a heat-stable, redox-active protein, contains an active site cysteine disulfide/dithiol. Oxidized thioredoxin, Trx-S, can be reduced to the dithiol form by NADPH and a specific flavoprotein enzyme, thioredoxin reductase. Reduced thioredoxin, Trx-(SH), participates in a number of redox reactions mostly linked to reduction of protein disulfides. Trx and thioredoxin reductase (TR), together with NADPH, form a redox complex in which TR catalyzes the electron transport from NADPH to Trx. The reduced thioredoxin then functions as an electron donor in a wide variety of different metabolic processes.
Disulfide-containing redox proteins not only facilitate disulfide formation, but also regulate and participate in a wide variety of physiological processes. The thioredoxin system serves, for example, as a hydrogen donor for ribonucleotide reductase and controls the activity of enzymes by redox reactions. Mammalian thioredoxin (MT) acts as a hydrogen donor for ribonucleotide reductase and methionine sulfoxide reductase, facilitates refolding of disulfide-containing proteins, and activates the glucocorticoid and interleukin-2 receptors. MT also modulates the DNA binding activity of some transcription factors either directly (TFIIIC, BZLF1, and NF-kB) or indirectly (AP-1) through the nuclear factor Ref-1. The importance of the redox regulation of transcription factors is exemplified by the v-fos oncogene where a point mutation of the thioredoxin-modulated cysteine residue results in constitutive activation of the AP-1 complex. Thioredoxin is secreted by cells using a leaderless pathway and stimulates the proliferation of lymphoid cells, fibroblasts, and a variety of human solid tumor cell lines. Furthermore, thioredoxin is an essential component of early pregnancy factor, inhibits human immunodeficiency virus expression in macrophages, reduces H2O2, scavenges free radicals, and protects cells against oxidative stress (Abate, C. et al., (1990) Science 249: 1157-1161; Rosen, A. et al. (1995) Int. Immunol. 7: 625-633; Tagaya, Y. et al (1989) EMBO J. 8: 757-764; Newman, G. W. (1994) J. Expt. Med. 180: 359-363; and Makino, Y. (1996) J. Clin. Invest. 98: 2469-2477).
Short-chain alcohol dehydrogenases (SCADs) are a family of dehydrogenases that share only 15% to 30% sequence identity, with similarity predominantly in the coenzyme binding domain and the substrate binding domain. In addition to the well-known role in detoxification of ethanol, SCADs are also involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders. For example, retinol dehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol. Chem. 270:1107-1112) that converts retinol to retinal, the precursor of retinoic acid. Retinoic acid, a regulator of differentiation and apoptosis, has been shown to down-regulate genes involved in cell proliferation and inflammation (Chai, X. et al. (1995) J. Biol. Chem. 270:3900-3904). In addition, retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et al. (1996) Genomics 36:424-430).
Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabolism (Weiss, B. (1991) Neurotoxicology 12:379-386; Collins, S. M. et al. (1992) Ann. N.Y. Acad. Sci. 664:415-424; Brown, J. K. and Imam, H. (1991) J. Inherit. Metab. Dis. 14:436-458). Many pathways of neurotransmitter metabolism require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as NAD+/NADH (Newsholme, E. A. and Leech, A. R. (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U.K. pp. 779-793). Degradation of catecholamines (epinephrine or norepinephrine) requires alcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (in peripheral tissue). NAD+-dependent aldehyde dehydrogenase oxidizes 5-hydroxyindole-3-acetate (the product of 5-hydroxytryptamine (serotonin) metabolism) in the brain, blood platelets, liver and pulmonary endothelium (Newsholme, E. A. and Leech, A. R. (supra) p. 786). Other neurotransmitter degradation pathways that utilize NAD+/NADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme, E. A. and Leech, A. R. supra. pp.790, 792). Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in a spectrum of disease states in different tissues including Parkinson""s disease and inherited myoclonus (McCance, K. L. and Huether, S. E. (1994) Pathophysiology, Mosby-Year Book, Inc., St. Louis, MO pp. 402-404; Gundlach, A. L. (1990) FASEB J. 4:2761-2766).
3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabolism produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands. Infants born with this deficiency typically die within 6 months (Watkins, P. et al. (1989) J. Clin. Invest. 83:771-777; Online Mendelian Inheritance in Man (OMIM), #261515). The neurodegeneration that is characteristic of Alzheimer""s disease involves development of extracellular plaques in certain brain regions. A major protein component of these plaques is the peptide amyloid-xcex2 (Axcex2), which is one of several cleavage products of amyloid precursor protein (APP). 3HACD has been shown to bind the Axcex2 peptide, and is overexpressed in neurons affected in Alzheimer""s disease. In addition, an antibody against 3HACD can block the toxic effects of Axcex2 in a cell culture model of Alzheimer""s disease (Yan, S. et al. (1997) Nature 389:689-695; OMIM, #602057).
17xcex2 hydroxysteroid dehydrogenase (17xcex2HSD6) plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone (DHTT). 17xcex2HSD6 acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3xcex1-diol, to androsterone which is readily glucuronidated and removed from tissues. 17xcex2HSD6 is active with both and estrogen and estrogen substrates when expressed in embryonic kidney 293 cells. At least five other isozymes of 17xcex2HSD have been identified that catalyze oxidation and/or reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M. G. and Russell, D. W. (1997) J. Biol. Chem. 272:15959-15966). For example, 17xcex2HSD1 preferentially reduces estradiol and is abundant in the ovary and placenta. 17xcex2HSD2 catalyzes oxidation of androgens and is present in the endometrium and placenta. 17xcex2HSD3 is exclusively a reductive enzyme in the testis (Geissler, W. M. et al. (1994) Nature Genet. 7:34-39). An excess of androgens such as DHTT can contribute to certain disease states such as benign prostatic hyperplasia and prostate cancer.
Steroids, such as estrogen, testosterone, corticosterone, and others, are generated from a common precursor, cholesterol, and are interconverted into one another. A wide variety of enzymes act upon cholesterol, including a number of dehydrogenases. One such dehydrogenase is 3-oxo-5-xcex1-steroid dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen-responsive tissues. OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen. Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia. A defect in OASD that prevents the conversion of testosterone into dihydrotestosterone leads to a rare form of male pseudohermaphroditis, characterized by defective formation of the external genitalia (Andersson, S., et al. (1991) Nature 354:159-161; Labrie, F., et al. (1992) Endocrinology 131:1571-1573; OMIM #264600). Thus, OASD plays a central role in sexual differentiation and androgen physiology.
The discovery of new oxidoreductase molecules and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative disorders including cancer, endocrine, metabolic, reproductive, neurological, autoimmune/inflammatory, and viral disorders.
The invention features substantially purified polypeptides, oxidoreductase molecules, referred to collectively as xe2x80x9cOXRExe2x80x9d and individually as xe2x80x9cOXRE-1,xe2x80x9d xe2x80x9cOXRE-2,xe2x80x9d xe2x80x9cOXRE-3,xe2x80x9d xe2x80x9cOXRE-4,xe2x80x9d xe2x80x9cOXRE-5,xe2x80x9d xe2x80x9cOXRE-6,xe2x80x9d xe2x80x9cOXRE-7,xe2x80x9d xe2x80x9cOXRE-8,xe2x80x9d xe2x80x9cOXRE-9,xe2x80x9d xe2x80x9cOXRE-10,xe2x80x9d xe2x80x9cOXRE-11,xe2x80x9d xe2x80x9cOXRE-12,xe2x80x9d xe2x80x9cOXRE-13,xe2x80x9d xe2x80x9cOXRE-14,xe2x80x9d and xe2x80x9cOXRE-15.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO:1-15 and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30 and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:1-15 and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of OXRE, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of OXRE, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 and fragments thereof.
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding OXRE.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of OXRE.
Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; a useful fragment of each nucleic acid; and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding OXRE were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze OXRE, along with applicable descriptions, references, and threshold parameters.